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. 2016 Feb 11;11(2):e0148213. doi: 10.1371/journal.pone.0148213

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© 2016 Cullen et al

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Fig 1 — (A). Schematic of the ADCK3 gene and its product. The relative position of exons/introns within the ADCK3 gene locus, the initiating ATG codon and the exons targeted by siRNA (ADCK3 siRNA #1 and #2) are depicted. Isoform 1 of ADCK3 is also shown with the relative positions of several important domains and motifs. Green rectangle: Region conserved amongst ADCK family members and specifically related to CoQ biosynthesis. Blue rectangle: Kinase-Like Domain (KLD) which contains 5 of the 12 prototypical kinase motifs involved in ATP binding and the phospohtransfer reaction (Blue ovals). Red rectangle: C-terminal region, whilst highly conserved amongst the ADCK3/4 subgroup, is divergent from that found in classical protein kinases and other ADCK family members. (B). ADCK3-FLAG localises to mitochondria. HeLa cells transiently transfected with pcDNA3.1/Hygro(+) only (Vec) or pcDNA3.1/Hygro(+) containing ADCK3 cDNA with either an N-terminal (pcDNA3.1/Hygro(+)-FLAG-ADCK3: FLAG-ADCK3) or C-terminal (pcDNA3.1/Hygro(+)-ADCK3-FLAG: ADCK3-FLAG) FLAG tag. Immunofluorescence performed with anti-FLAG and Alexa488-conjugated secondary antibodies (FLAG). Counterstaining for mitochondria was performed with Mitotracker Deep Red (Mitotracker). Nuclei were stained with Hoechst 3342. White bars: 15 μm. 63x mag. A single z-axis position is shown. See S1 Fig for images over multiple z-axis positions with ADCK3-EGFP. (C, D). Endogenous ADCK3 localises to mitochondria. Immunofluorescence (C) based analysis of HeLa cells conducted with anti-ADCK3 and Alexa488-conjugated secondary antibodies (ADCK3). HeLa cells treated with control siRNA (siRNA CON) or ADCK3 siRNA #1 and #2 for 48 h. Again, counterstaining for mitochondria was performed with Mitotracker Deep Red (Mitotracker). Nuclei were stained with Hoechst 3342. White bar: 15 μm. Arrow head indicates the position of significant perinuclear or ‘golgi-like’ staining. 63x mag. Cellular subfractionation of HeLa cells (D) was also performed. WCE, cytoplasmic (Cyto) and mitochondrial (Mito) fractions (20 μg) were separated via SDS-PAGE and immunoblotting conducted with antibodies to ADCK3, Tom20 (a mitochondrial marker) and α Tubulin (a cytosolic marker). (E). ADCK3-FLAG localises to mitochondrial cristae. Anti-FLAG immunogold electron microscopy performed with HeLa cells transiently transfected with vector expressing ADCK3-FLAG. Arrow Heads: OMM; Arrows: ADCK3-FLAG on cristae. (F, G). Endogenous ADCK3 associates with the matrix side of the inner mitochondrial membrane. Proteinase K protection (E) and Sonication/Alkaline extraction experiments (F) were conducted with crude mitochondria. Immunoblotting of samples/fractions separated via SDS-PAGE was performed with antibodies to ADCK3, Hsp60 (matrix protein), Tim23 (integral IMM protein facing the IMS), Cyt C (IMM associated IMS protein) and NDUFB6 (integral IMM protein). PK: proteinase K. S: supernatant fraction. P: pellet fraction.